Caracterización de nuevos reguladores que determinen el posicionamiento y la formación de células madre en arabidopsis thaliana

Abstract

ABSTRACT Plants have the capacity of grow and develop new tissues and organs during all their life. This capacity required the generation and activity of new stem cells. Although the activity of these stem cells has been studied the molecular mechanism that produce the formation of new stem cells are not well known. The main objective in this thesis has been studied these mechanism using the root of the model plant Arabidopsis thaliana. We have developed two different approaches, by one hand we have identified genes involved in keep and lead the cellular reprogramation to generate stem cells using a bioinformatics strategy. This approach has identified genes with specific or enriched expression in the pericycle, it is this tissue that is reprogrammed to generate new stem cells of endogenous lateral roots and also that can be reprogrammed by the hormonal supplementation and in vitro culture to form proliferating of cell masses or callus. This result in a list of about 300 genes, which we classify by functional categories, focuses on the analysis of transcription factors (27), because these could be involved in maintaining the specific expression in the pericycle and are usually key elements in the signaling. Using loss-of-function lines of these factors were performed studies that analyzed the capacity of lateral root and callus formation to see which genes affected cell pluripotency. This analysis identifies two regulatory factors of pluripotency during the post-embryonic development, FUF1 and OBP4. The subsequent study of the OBP4 factor determined that its overexpression initiated the proliferation of the pericycle, presenting more than one layer. In addition, overexpression of OBP4 in callus-forming experiments promotes cell proliferation causing an increase in the weight of the callus formed. We also characterize its effect on alf4-1 mutant, an affected mutant in callus formation, noting that overexpression of OBP4 is also capable of enhancing callus formation in the mutant alf4-1. The second approach that has been followed in this thesis to identify regulators of the formation of new stem cells has consisted in the generation of a library of mutants with Ethyl methyl sulphonate (EMS) and in the subsequent identification of affected mutants in the early stages of lateral root formation according to markers pSCARECROW:GFP and pWUSCHEL-RELATED-HOMEOBOX5:YFP. This search resulted in the localization of two mutants that affected the formation of stem cells in root, which caused that they did not form lateral roots. The first mutant that we identify is a recessive mutant of loss of function affecting the GNOM gene and which we call gnom-204. The data provided by the DR5 marker indicated a lack of expression in the root zone known as the oscillation zone and an excess of marking in the maturation zone indicative of ectopic formation of pre-branching sites. Pre-branching sites are the first cell specification process associated with the formation of pluripotent stem cells or lateral root founding cells and depend on the so-called oscillating periodic expression of genes in the oscillation zone. It has been described that the function of GNOM is related to the regulation of vesicular traffic mediated by the trans-golgi network, a function that is necessary for the activity of auxin transporters. The identified mutation could allow us to analyze the relationship between auxin trafficking and oscillations in future experiments, identifying new functions of GNOM in the specification of stem cells. The second mutant that we identify, potent, affects the IAA18 gene causing a dominant mutation, similar to others described mutants that affect other IAA by preventing its degradation induce by auxin and the lack of signaling of this hormone. Our results show that this mutation causes the continuous activation of the oscillations in phase, which are the ones that determine the specification of pre-branch sites and founder cells coincident with their maximum expression. This over-activation of oscillations in potent generates the continuous specification of pre-branch sites and founding cells along the root. In addition, these founding cells are blocked at that stage of development. Treatments with the hormone auxin, which do not cause reprogramming of the pericycle cells but which stimulate the development of founding cells, are able to get these cells to pass this blockage which results in the over-production of lateral roots in the mutant. One of the factors described that interact with IAA18 is AUXIN RESPONSE FACTOR (ARF) 7 which is an oscillating factor in anti-phase, which means that its expression is displaced in time a period with respect to the genes in phase. Our results indicate that its loss of function mutant has over-activated phase oscillations, as in the case of potent mutant, which also results in the over-production of founder cells. AUX/IAA factors have been reported to act as transcriptional repressors of ARF. In this thesis, the knowledge of the mechanism of regulation of oscillations mediated by IAA18 and ARF7 downstream of auxins has been deepened. We observed that the interaction between IAA18 and potent with ARF7 is dependent on the concentration of auxins in the medium, which would occur through direct interaction with the hormone. The dynamics of the interaction after auxin application occurs similarly to the activation of the expression of the oscillating gene in phase LOB-DOMAIN16, which is also a direct target of ARF7. Our data suggest that auxins would act in two different ways, first, at low concentrations (10 nM) it would act as inducers of the interaction between both proteins relatively quickly during the first hour of exposure, then to cause degradation of IAA18 at 3 hours, which make free ARF7 from inhibition of IAA18. In potent we also observed that ARF7 protein levels are repressed while auxin levels have increased, suggesting the existence of a feedback loop between the heterodimer ARF7-IAA18 and auxins and ARF7 that could be responsible for maintaining the rhythm of the oscillations genes in phase. Although the levels of the IAA18-ARF7 interaction turn out to be very low and make it difficult to study we can see periodic increases that coincide with the estimated time of the oscillations. Secondly higher amounts of auxins (1M) would cause degradation of IAA18 observably in the first hour but at the same time its transcriptional activation, so that an increase of the activation in the first hour is detected, also coinciding with the activation transcription of LBD16. In addition, the inactivation of IAA18 by CRISPR-CAS9 technology results in plants with a smaller number of lateral roots, which supports the role of IAA18 as a regulator of oscillating gene expression, important for the positioning of lateral roots. Also, the microscopic analysis of the founder cells observed in potent shows variability of cells sizes, which indicate that it appear to undergo cell divisions. The analysis of markers shows that these divisions tend to be symmetric in size and affect the polarization of proteins normally observed in the divisions of the founder cells. Thus, the MAKR4 protein, which is located on the membrane between two founder cells, was found in the mutant located on both sides of the membrane. Finally, potent presents the formation of other types of post-embryonic roots such as adventitious roots, while the mutant arf7-1 is not affected in this process, which could indicate that IAA18 acts in the formation of all types of roots but would do so through interaction with distinct ARFs.